Double-stranded RNA molecules (dsRNA) can block gene expression by virtue of a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNA III Dicer enzyme processes dsRNA into small interfering RNA (also sometimes called short interfering RNA or siRNA) of approximately 22 nucleotides. One strand of the siRNA (the “antisense strand”) then serves as a guide sequence to induce cleavage of messenger RNAs (mRNAs) including a nucleotide sequence which is at least partially complementary to the sequence of the antisense strand by an RNA-induced silencing complex RISC. The antisense strand is not cleaved or otherwise degraded in this process, and the RISC including the antisense strand can subsequently affect the cleavage of further mRNAs.
The process of posttranscriptional dsRNA-dependent gene silencing is commonly referred to as RNA interference (RNAi) (Tuschl, T. Chembiochem 2, 239-45 (2001), Zamore, P. D. Science 296, 1265-9 (2002), Hannon, G. J. Nature 418, 244-51 (2002)). It has been proposed that eukaryotes utilize RNAi to protect their genomes against invading foreign genetic elements such as viruses. The formation of dsRNA during viral replication is interpreted by the cell as a signal for unwanted gene activity (Ahlquist, P. Science 296, 1270-3 (2002), Plasterk, R. H. Science 296, 1263-5 (2002)). Dicer RNase III rapidly processes dsRNA to small dsRNA fragments of distinct size and structure, the small interfering RNAs (siRNAs), which direct the sequence-specific degradation of the single-stranded mRNAs of the invading genes (Elbashir, S. M. et al. Nature 411, 494-8 (2001), Elbashir, S. M., et al. Genes Dev 15, 188-200 (2001), Hammond, S. M., et al. Nature 404, 293-6 (2000), Zamore, P. D., et al. Cell 101, 25-33 (2000)). Such siRNA duplexes have 2-3 nt 3′ overhanging ends and contain 5′ phosphate and free 3′ hydroxyl termini (Elbashir, S. M., et al. Embo J 20, 6877-88 (2001)). Cellular delivery of synthetic siRNA duplexes or introduction of siRNA by plasmids or viral vectors is now widely used to disrupt the activity of cellular genes homologous in sequence to the introduced dsRNA.
An understanding of how siRNAs interact with mammalian systems is important for refining this gene silencing technology and for developing gene-specific therapeutic agents (Tuschl, T. et al. Mol Interv 2, 158-67 (2002)). For the recognition of long dsRNA two different detection modes are known, the serine threonine kinase PKR (Williams, B. R. Sci Signal Transduction Knowledge Enviroment 89, RE2 (2001), Meurs, E. F. et al. Virol 66, 5805-14 (1992), Katze, M. G. et al. Mol Cell Biol 11, 5497-505 (1991)) and TLR3 (Alexopoulou, L., et al. Nature 413, 732-8 (2001)). While PKR is located in the cytosol, TLR3 is present in the endosomal compartment (Matsumoto, M. et al. J Immunol 171, 3154-62 (2003)). TLR3 is a member of the Toll-like receptor family that has evolved to detect pathogen-specific molecules (Takeda, K., et al. Annu Rev Immunol 21, 335-76 (2003)).
PKR possesses two dsRNA-binding domains, one of which has high affinity for dsRNA, while the other shows considerably lower affinity. Full activation of the PKR-mediated response requires simultaneous binding of dsRNA to both domains, which may be facilitated by long dsRNAs, e.g. dsRNAs exceeding 50-80 nucleotide pairs in length, and seems to require dimerization (Manche, L., et al., Mol Cell Biol, 12, 5238-48 (1992); Williams, B. G., Oncogene, 18, 6112-20 (1999)). High concentrations of dsRNAs, including dsRNAs of less than 50 nucleotide pairs, or of other ligands for the dsRNA binding site (e.g. Alu RNAs) inhibit the activation of PKR. Early invenstigations seemed to prove that siRNA duplexes are short enough to bypass general dsRNA-induced unspecific effects in vertebrate cells (Bitko, V. et al. BMC Microbiol, 1, 34 (2001)). A number of more recent publications, however, indicates that a large array of genes is differentially regulated upon the introduction of short dsRNAs, including genes involved in the interferon pathway and specifically the activation of PKR, even if not to a similar extent as compared to the effect of long dsRNAs (Jackson, A. L. and Linsley, P. S., Trends Genet, 20, 521-4 (2004); Jackson, A. L., et al., Nat Biotechnol, 21, 635-7 (2003); Moss, E. G., and Taylor, J. M., Nat Cell Biol, 5, 771-2 (2003); Bridge, A. J., et al., Nat Genet, 34, 263-4 (2003); Sledz, C. A., et al., Nat Cell Biol, 5, 834-9 (2003); Heidel, J. D., et al., Nat Biotechnol, 22, 1579-81 (2004); Kim, D. H., et al., Nat Biotechnol, 22, 321-5 (2004); Zheng, X., and Bevilacqua, P. C., RNA, 10, 1934-45 (2004); Pebernard, S., and Iggo, R., Differentiation, 72, 103-11 (2004)). Which genes are up- or downregulated seems to be at least partly siRNA-sequence specific, and the mechanism, or mechanisms, underlying this regulation remain(s) to be elucidated.
A second characteristic feature of viral nucleic acids used by the immune system to recognize viral infection are CpG motifs found in viral DNA, which are detected via TLR9 (Lund, J., et al. J Exp Med 198, 513-520 (2003), Krug, A. et al. Blood 103, 1433-7 (2004)). CpG motifs are unmethylated CG dinucleotides with certain flanking bases. The frequency of CpG motifs is suppressed in vertebrates, allowing the vertebrate immune system to detect microbial DNA based on such CpG motifs (Krieg, A. M. et al. Nature 374, 546-9 (1995), Bauer, S. et al. Proc Natl Acad Sci USA 98, 9237-42 (2001), Wagner, H. Curr Opin Microbiol 5, 62-9 (2002)). Like TLR3, TLR9 is located in the endosomal compartment where it directly binds to CpG motifs (Latz, E. et al. Nat Immunol 5, 190-8 (2004)).
In addition to long dsRNA and CpG DNA, two recent publications suggest a third mechanism by which viral nucleic acids are recognized. These studies demonstrate that single-stranded RNA (ssRNA) of ssRNA viruses is detected via TLR7 (mouse and human) and TLR8 (only human) (Diebold, S. S., Science 303, 1529-31 (2004), Heil, F. et al. Science 303, 1526-9 (2004)). Guanine analogues have been identified earlier as specific ligands for TLR7 and TLR8 (Lee, J. et al. Proc Natl Acad Sci USA 100, 6646-51 (2003), Heil, F. et al. Eur J Immunol 33, 2987-97 (2003)). Like TLR9 (receptor for CpG DNA)(Latz, E. et al. Nat Immunol 5, 190-8 (2004)), TLR7 and TLR8 are located in the endosomal membrane.
Detection of viral nucleic acids leads to the production of type I IFN (IFN-α and IFN-β). The major producer of type I IFN in humans is the plasmacytoid dendritic cell (also called interferon producing cell, IPC). The plasmacytoid dendritic cell (PDC) is a highly specialized subset of dendritic cells that is thought to function as a sentinel for viral infection and that is responsible for the vast amount of type I IFN during viral infection (Asselin-Paturel, C. et al. Nat Immunol 2, 1144-50 (2001)). There is increasing evidence that PDC preferentially use nucleic acid-based molecular patterns to detect viral infection. TLR expression of human and mouse PDC is limited to TLR7 and TLR9 (Krug, A. et al. Eur J Immunol 31, 3026-37 (2001), Hornung, V. et al. J Immunol 168, 4531-7 (2002), Edwards, A. D. et al. Eur J Immunol 33, 827-33 (2003)).
Tokunaga et al, J. Natl. Cancer Inst. 72:955-962 (1984); Messina et al., J. Immunol. 147: 1759-1764 (1991); Krieg et al., Nature 374: 546-549 (1995); Sato et al, Science 273: 352-354 (1996), teach that the presence of CpG dinucleotides in certain sequence contexts in bacterial and synthetic oligodeoxyribonucleotides (CpG DNAs) are known to activate vertebrate innate immune reaction, T-cells and B cells.
Yamamoto et al., Jpn. J. Cancer Res. 79: 866-873 (1988); Halpern et al., Cell Immunol., 167: 72-78 (1996); Klinman et al., Proc. Natl. Acad. Sci. U.S.A. 93: 2879-2883 (1996); Zhao et al., Antisense Nucleic Acid Drug Dev. 7: 495-502 (1997) teach that the activation of immune cells by CpG DNA induces secretion of a number of cytokines, including IFN-.gamma., IL-12, TNF-.alpha., and IL-6, and stimulates expression of costimulatory surface molecules.
Krieg et al., supra; Yamamoto et al, J. Immunol. 148; 4072-4076 (1992); Tokunaga et al., Microbiol. Immunol. 36: 55-66 (1992); Liang et al., J. Clin. Invest. 98: 1119-1129 (1996); Hartmann et al., J. Immunol. 164: 1617-1624 (2000), teach that the presence of a CpG dinucleotide and the sequences flanking the dinucleotide play a critical role in determining the immunostimulatory activity of DNA, that CpG dinucleotides in palindromic or non-palindromic hexameric sequences (P1. P2CGP3P4) are required for immune stimulation, and further, that PuPuCGPyPy and PuTCG motifs optimally activate murine and human immune systems, respectively.
While these findings demonstrate that oligonucleotides are useful as immune stimulating agents, some problems with such use still exist. For example, long oligonucleotides are expensive to make and species specificity of flanking sequences limits the breadth of utility of any given oligonucleotide. There is, therefore, a need for less expensive immunostimulatory agents, and preferably immunostimulatory agents that have cross-species efficacy as well as a need to identify additional sequence specific motifs.